Vectorization of dsRNA by cationic particles and topical use

ABSTRACT

The present invention relates to vectorization of double stranded RNA oligonucleotides by cationic particles chosen from among surfactant micelles, block or non-block polymer micelles, cationic liposomes and niosomes, cationic oleosomes and cationic nanoemulsions, as well as from among cationic organic or inorganic particles and nanocapsules, and topical compositions for the skin, mucous membranes or eyes.

REFERENCE TO PRIOR APPLICATIONS

This application claims priority to U.S. provisional application60/689,529 filed Jun. 13, 2005, and to French patent application 0551301filed May 19, 2005, both incorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to vectorization of double stranded RNAoligonucleotides by cationic particles chosen from among surfactantmicelles, block or non-block polymer micelles, cationic liposomes andniosomes, cationic oleosomes and cationic nanoemulsions, as well as fromamong cationic organic or inorganic particles and nanocapsules, andtopical compositions for the skin, mucous membranes or eyes.

BACKGROUND OF THE INVENTION

Research is being conducted on the use of substances having specificactivity, such as specific biological activity, in the cosmetics fieldsuch as skin care or hair care, but also in the dermatological andpharmaceutical fields.

Recently, the use of dsRNA and more particularly siRNA (containing 12 to40 nucleotides) has made it possible to achieve specific activity ininhibition of the synthesis of a target protein. The molecular mechanismtaking place involves double stranded RNA fragments composed of 12 to 40nucleotides. Degradation of the target mRNA is achieved by activation ofthe complex known as RISC (RNA Induced Silencing Complex), which acts byfixation of the anti-sense strand of the dsRNA onto the mRNA. Thesedouble stranded RNA oligonucleotides are also known as dsRNA or elsesiRNA (for “short interfering” RNA); see Tuschl T., Chem. Biochem. 2001,2, 239-245; Nykanen A et al., Cell 2001, 107, 309-321, Dorsett Y.,Nature, April 2004, Vol. 3, pages 318-329; and Downward, J., BMJ 2004,328, 1245-1248.

However, the topical application of these siRNA, which can havemolecular weights of approximately 15 to 17 kD, gives rise to theproblem of penetration. In fact, for the specific activity relative tothe selected siRNA to be effective, it must penetrate into the targetcell (such as the keratinocytes or the melanocytes, etc.) as far as itscytoplasm. As it happens, the stratum corneum, the site of the skinbarrier function, is difficult to penetrate.

International Application WO 03/101376 describes cosmetic preparationscomprising at least one double stranded RNA oligonucleotide. In thispublication, the oligonucleotide is necessarily complexed with acationic polymer such as PEI or chitosan, after which this complex ofoligonucleotide and cationic polymer can be encapsulated in liposomes orniosomes and/or adsorbed at the surface of particles such as liposomes,niosomes, oleosomes, nanospheres and nanocapsules. Therefore, the dsRNAis necessarily complexed with a cationic polymer before being associatedwith a type of particle. In addition, the surfactant micelles are notdescribed as an efficient medicinal solution for vectorization of siRNA.

In the article “Intercellular adhesion molecule-1 suppression in skin bytopical delivery of anti-sense oligonucleotides,” Mehta et al. (J.Invest. Dermatol. 115, 805-812, 2000), the authors transport singlestranded RNA in an emulsion containing 25% of surfactants (10% ofglyceryl stearate and 15% of 40 OE stearate). Besides the fact that thisarticle does not describe the siRNA, the proposed medicinal solution isnot compatible with good tolerance. In fact, the 25% of surfactants willhave the effect of destroying the barrier function of the skin, thusincreasing its permeability to external elements and dramaticallyfavoring its dehydration: results that are not sought in the fields ofapplication envisioned by the present invention.

In the publication “Lipidic carriers of siRNA: differences in theformulation, cell uptake and delivery with plasmid DNA,” Spagnou et al.(Biochemistry, 43, 13348-56, 2004), the authors use cationic liposomessuch as lipofectamine 2000 or DOPE liposomes “cationized” withcholesteryloxycarbonyl-3,7-diazanone-1,9-diamine (CDAN). For safetyreasons, lipofectamine cannot be used on human skin.

Other publications (Yano Junichi et al., Clinical Cancer Research, 2004,Spagnou et al., Biochemistry, 2004, and Ma Zheng et al., Biochemical andBiophysical Research Communications, 2004) have proposed associatingsiRNA with cationic vesicles composed of oleyl chain lipids containingunsaturated bonds that make them very sensitive to oxidation, andphospholipids, these vesicles being intended to be used immediatelyafter preparation, because they are not very stable in time.

International Patent Application WO 2004/046354 describes the use ofsiRNA for cutaneous dysfunction, preferably at the dermis level. Thecompositions described, such as PIT and W/O, O/W and W/O/W emulsions,are all nonionic. In that patent application, no appraisal is made ofthe major and essential interest of using cationic surfactants incationic particles in order to favor penetration of the siRNA intocutaneous structures. Moreover, although the PIT can be smaller than μmsize, they are necessarily nonionic, as described in InternationalPatent WO96/28132.

International Application WO 03/106636 describes the association of apolynucleotide with a cationic surfactant such as cetyltriammoniumchloride to form a complex. It is then necessary to stabilize thiscomplex by an incubation step at a temperature of between 35 and 50° C.In a second step, it is possible to dehydrate the mixture to obtaintherefrom a powder, which will then be diluted in a chosen solventbefore use (injection). Once the complex has been formed, it is alsopossible to stabilize it by addition of an amphiphilic lipid, which canbe a nonionic surfactant. This ternary association is then dehydrated toobtain a powder, which will then be diluted in an appropriate aqueoussolution.

International Application WO 03/106636 does not specify that theconcentration of the cationic surfactant must be above the CMC (criticalmicellar concentration) in order to form the complex of polynucleotideand cationic surfactant, since the complex can be formed below the CMC.It is only once this complex has been formed that the addition ofanother amphiphilic compound capable of forming micelles is envisioned.In this way the integration of a complex of polynucleotide and cationicsurfactant into amphiphilic micelles is achieved. Thus the question ofassociating cationic particles with a polynucleotide is not addressed inthat document.

However, there remains a need to identify a way to permit thepenetration of siRNA into the skin models.

SUMMARY OF THE INVENTION

Accordingly, one object of the present invention is to providecompositions and methods for permitting the penetration ofdouble-stranded and/or siRNA into keratinous and/or ocular substances.

The invention provides a topical or ocular composition comprising atleast one dsRNA associated with at least one cationic particle of a sizeless than or equal to 1 μm and having a zeta potential of from 10 to 80mV, and which is chosen from a surfactant micelle, a block polymermicelle, a liposome of nonionic and/or cationic surfactants, niosomes,oleosomes, particles of nanoemulsions, nanocapsules, organic particles,or inorganic particles.

The invention also provides providing a RNA molecule to a keratinousand/or ocular surface for a cosmetic purpose using the compositiondescribed herein.

The invention also provides methods of inhibiting a protein in akeratinous substance, e.g., skin and hair, and/or eyes by administeringthe composition as well as generally methods of delivering the dsRNA toan individual in need thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

A more complete appreciation of the invention and many of the attendantadvantages thereof will be readily obtained as the same becomes betterunderstood by reference to the following detailed description whenconsidered in connection with the accompanying drawings, wherein:

Figure I shows the results of cell targeting in Episkin—day 6 where Fig.I-1 shows Block-It Fluorescent Oligo (DAPI); Fig. I-2 shows Block-ItFluorescent Oligo+Lipofectamine 2000 (DAPI); Fig. I-3 shows Block-ItFluorescent Oligo+E2 (×40, DAPI); Fig. I-4: E2 (×40, DAPI); Fig. I-5shows Block-It Fluorescent Oligo+E2 (×100, DAPI); and Fig. I-6 showsBlock-It Fluorescent Oligo+E2 (×100).

Figure II shows the results of Cell targeting in Episkin—day 13 whereFig. II-1 shows Block-It Fluorescent Oligo (×40, DAPI); and Figs. IIIAand IIIB show visualization of the effect of the formulation comprisingoctyl glucoside and CTAB in different concentrations on the viability ofHaCaT cells.

DETAILED DESCRIPTION OF THE INVENTION

Unexpectedly, the association of siRNA with cationic particles permits avery significant improvement for penetrating into target cells of atissue such as the skin, especially skin models. After penetration, thesiRNA becomes active. The penetration can be evaluated using afluorescent marker fixed on the siRNA, and its activity can be evaluatedby quantification of the targeted messenger by quantitative PCR or byassay of the protein corresponding to the targeted messenger RNA. Thecationic particles of the invention can be surfactant micelles, block ornon-block polymer micelles, cationic liposomes and niosomes, cationicoleosomes, cationic nanoemulsions, as well as cationic organic orinorganic particles and nanocapsules.

Thus, the object of the present invention therefore relates firstly to atopical or ocular composition comprising at least one double strandedRNA oligonucleotide, characterized in that the double stranded RNAoligonucleotide is associated with at least one cationic particle ofsize smaller than or equal to 1 μm, of zeta potential between 10 and 80mV, chosen from among surfactant micelles, in particular micelles ofnonionic amphiphilic surfactants and cationic surfactants, block polymermicelles, in particular micelles of cationic amphiphilic block polymer,micelles of nonionic amphiphilic block polymer and of cationicamphiphilic block polymer and micelles of nonionic amphiphilic blockpolymer and of cationic surfactant, liposomes of nonionic and cationicsurfactants, niosomes, oleosomes, particles of nanoemulsions,nanocapsules, organic particles or inorganic particles.

The present invention relates to the association of cationic particleswith at least one dsRNA that will adhere to the surface of the particle,the latter acting as a vehicle to allow it to penetrate into thecutaneous structures and into the target cells of the skin, mucousmembranes or even eyes, for cosmetic, dermatological and/or ophthalmicapplications.

The cationic particles according to the invention are particles having asize less than or equal to 1 μm, preferably less than or equal to 500nm, even more preferentially less than or equal to 300 nm, which can bemeasured with, for example, a laser granulometer of the type BI90 plusof the Brookhaven Co., and which have a zeta potential of between 10 and80 mV, measurable with a zetameter of the type DELSA 440 of theCoultronics Co.

Hereinafter there is presented a non-exhaustive list of cationicparticles than can be used according to the invention.

Surfactant Micelles

As known in the art, micelles are aggregates that amphiphilic moleculesform spontaneously when they are solubilized in water or oil above acertain critical concentration: the CMC.

The micelles that can be used within the scope of the invention arecomposed of at least one cationic surfactant. This cationic surfactantcan be associated with one or more nonionic amphiphilic surfactants.

The person skilled in the art will advantageously select the nonionicand cationic surfactants in the 1998 and subsequent editions ofMcCutcheon's “Emulsifiers and Detergents”.

Non-limiting examples of the cationic surfactants that can be usedwithin the scope of the invention are listed hereinafter.

Non-limiting examples of nonionic surfactants that can be used are:alkyl and polyalkyl (C6 to C30, saturated or unsaturated, branched orlinear) esters or ethers of POE, glycerol and polyglycerol, of sorbitanwith or without oxyethylene groups, of sucrose, of glucose with orwithout oxyethylene groups, of nialtose and of POP-POE. If mixtures ofnonionic surfactants and cationic surfactants are included, theirrespective proportions by weight will be between 99/1 and 1/99.

The proportions of surfactants forming the micelles will be dependent onthe CMC thereof. Within the scope of the invention, however, theconcentration of micellar surfactants will be between 0.1 and 10% andpreferably between 0.2 and 5% by weight relative to the total weight ofthe composition.

Block Polymer Micelles

The micelles of amphiphilic block polymers can be prepared by the methoddescribed in International Application WO 04/035013.

The block copolymers that are useful for preparation of micellesassociated with the dsRNA according to the invention are, in particular,amphiphilic block polymers, preferably of nonionic, di-block ortri-block type that can form micelles upon contact with water. Inparticular, they are of the di-block type (A-B) or of the tri-block type(A-B-A), where A corresponds to a nonionic hydrophilic polymeric blockand B to a hydrophobic polymeric block. The molecular weight of thepolymers can be between 1000 and 100,000, and the A/B ratio can bebetween 1/100 and 50/1.

The nonionic hydrophilic polymeric block can be chosen from amongethylene polyoxide (POE), polyvinylpyrrolidone (PVP) and polyacrylicacid (PAA).

The hydrophobic polymeric block can be chosen from among polystyrene,poly(tert-butylstyrene), poly(methyl methacrylate), poly(ethylacrylate), poly(butyl acrylate), poly(butyl methacrylate), poly(vinylacetate), polycaprolactones, polycaprolactams, polydimethylsiloxanes,polyoxides of C3 to C6 alkylene, poly(aspartic acid), poly(lactic acid),poly(glycolic acid), polyleucine, polybutadienes, polyethylenes,polypropylenes and polybutylenes.

The block copolymer is preferably chosen from among the following blockcopolymers:

propylene polyoxide/ethylene polyoxide

polystyrene/polyoxyethylene

polymethyl methacrylate/polyoxyethylene

polybutyl methacrylate/polyoxyethylene

polyoxybutylene/polyoxyethylene

polycaprolactone/polyoxyethylene

polyethylene/polyoxyethylene

polyoxyethylene/polyoxybutylene/polyoxyethylene.

Within the scope of the invention, the following can be added to add tothe micellar composition:

a cationic amphiphilic block polymer in which one of the blocks iscationic and can be chosen from among, for example:

trimethylethylammonium polymethacrylate;

quaternized dimethylaminoethyl polymethacrylate;

polymethylvinylimidazolium;

polyvinylbenzyltrimethylammonium chloride;

the association of a nonionic amphiphilic block polymer with a cationicamphiphilic block polymer is such that the ratio between the two will bebetween 99/1 and 1/99; and/or

at least one cationic surfactant such as listed hereinafter.

In this case, the respective ratio between the nonionic amphiphilicblock polymer and the cationic surfactant will be between 50/50 and99/1.

Within the scope of the invention, the concentration of micellar blockpolymers that are or are not associated with a cationic surfactant willbe between 0.1 and 10% and preferably between 0.2 and 5% by weightrelative to the total weight of the composition.

In an alternative version of the invention, it is also possible to formmicelles composed of cationic amphiphilic block polymers such asdescribed in the foregoing.

Liposomes and Niosomes

The nonionic amphiphilic lipids capable of forming nonionic liposomesare in particular those described in Patent Application EP 0582503.

In particular, the nonionic amphiphilic lipids can be composed of amixture of esters of at least one polyol chosen in the group formed bypolyethylene glycol having 1 to 60 ethylene oxide units, sorbitan,sorbitan with 2 to 60 ethylene oxide units, glycerol with 2 to 30ethylene oxide units, polyglycerols with 2 to 15 glycerol units,sucroses, glucoses with 2 to 30 ethylene oxide units, and at least onefatty acid having a CS to C17 saturated or unsaturated, linear orbranched alkyl chain, the number of alkyl chains per polyol group beingbetween 1 and 10.

The expression “mixture of esters” covers not only mixtures of pureesters of different chemical families but also products that contains aplurality of chemically pure polyol esters of the same family invariable proportions. This is the case in particular of products thathave a statistical formula in their hydrophilic part, such as apolyglycerol ester of formula CO—(OCH₂—CHOH—CH₂)_(n)—OH, where n is astatistical value, and that can contain diverse proportions of estersfor which n=1, n=2, n=3, n=4, etc.; this is also the case of esterscontaining a plurality of alkyl chains in their lipophilic part, such asthe cocoates, which contain C5 to C17 alkyl chains, or the isostearates,where the C17 alkyl chains are a complex mixture of isomeric forms; itis also the case of products composed of mixtures of mono-, di-, tri- orpolyesters of one and the same polyol. It must be noted that a productthat would contain only a single ester capable of forming vesiclestogether with impurities of another type could not be used according tothe invention.

Commercial esters that can be used alone according to the invention,because in reality they are mixtures of esters are, for example, thefollowing:

-   -   the partial esters of sorbitan (or sorbitol anhydride) and fatty        acid, sold under the trade names “SPAN 20, 40, 60 and 80” by the        ICI Co.;    -   sorbitan isostearate, sold under the trade name “SI 10 R NIKKOL”        by the NIKKO Co.;    -   sorbitan stearate with 4 ethylene oxide units, sold under the        name “TWEEN 61” by the ICI Co.;    -   polyethylene glycol stearate with 8 ethylene oxide units, sold        under the name “MYR J 45” by the ICI Co.;    -   polyethylene glycol monostearate of formula EMI6.1, in which        formula n is equal to 4, sold under the name “MYS 4” by the        NIKKO Co.;    -   polyethylene glycol stearate of molecular weight 400, chemical        grade or grade produced by biotechnology, sold by the UNICHEMA        Co.;    -   diglyceryl stearate with 4 ethylene oxide units, sold under the        name “HOSTACERINE DGS” by the HOECHST Co.;    -   tetraglycerol stearate, sold under the name “TETRAGLYN 1S” by        the NIKKO Co.;    -   diglyceryl isostearate, sold by the SOLVAY Co.;    -   diglyceryl distearate, sold under the name “EMALEX DSG 2” by the        NIHON Co.;    -   sucrose mono-, di- and tripalmitostearates, sold under the names        “F50, F70, F110 AND F160 CRODESTA” by the CRODA Co.;    -   the mixture of sucrose mono- and dipalmitostearates, sold under        the name “GRILLOTEN PSE 141 G” by the GRILLO Co.;    -   the mixture of sucrose stearate and sucrose cocoate, sold under        the name “ARLATONE 2121” by the ICI Co.;    -   methylglucose distearate with 20 ethylene oxide units, sold        under the name ‘GLUCAM E 20 DISTEARATE” by the AMERCHOL Co.

Of course, it is possible to use mixed combinations of these differentproducts that are already mixtures or mixed combinations of theseproducts with pure products.

The cationic surfactants are chosen in the list hereinafter, in such away that they impart a pH of between 5 and 8 to the dispersion, whereinthe ratio by weight between the quantity of nonionic amphiphilic lipidsand the quantity of cationic surfactants in the lipid phase is between50/1 and 50/25, and the ratio by weight between the lipid phase and theaqueous dispersion phase is between 1/1,000 and 300/1,000.

Oleosomes

The oleosomes relating to the invention are described in PatentApplication EP

They comprise an emulsion of the oil-in-water type formed from oilglobules provided with a lamellar liquid crystal coating and dispersedin an aqueous phase, characterized in that each oil globule isindividually coated with a monolamellar or oligolamellar layer (1 to 10lamellas that are visible under the transmission electron microscopeafter cryofracture) obtained from at least one lipophilic surface-activeagent, at least one hydrophilic surface-active agent and at least onecationic surfactant, which imparts a pH ranging from 5 to 8 to theemulsion, the coated oil globules having an average diameter less than500 nanometers.

The lipophilic surface-active agent and the hydrophilic surface-activeagent each contain at least one saturated fatty chain having more thanapproximately 12 carbon atoms. More preferentially, this fatty chaincontains 16 to 22 carbon atoms.

According to another preferential embodiment of the invention, thelipophilic surface-active agent has an HLB of between approximately 2and approximately 5. As is well known, there is understood by HLB(hydrophilic-lipophilic balance) the equilibrium between the dimensionand strength of the hydrophilic group and the dimension and strength ofthe lipophilic group of the surface-active agent.

Examples of such lipophilic surface-active agents are sucrosedistearate, diglyceryl distearate, tetraglyceryl tristearate,decaglyceryl decastearate, diglyceryl monostearate, hexaglyceryltristearate, decaglyceryl pentastearate, sorbitan monostearate, sorbitantristearate, diethylene glycol monostearate, the glycerol ester ofpalmitic and stearic acids, 2 OE polyoxyethylene monostearate(containing 2 oxyethylene units), glyceryl mono- and dibehenate, andpentaerythritol tetrastearate.

The hydrophilic surface-active agent preferably has an HLB of betweenapproximately 8 and approximately 12.

As examples of such hydrophilic surface-active agents there can be citedthe following compounds: polyoxyethylene (4 OE) sorbitan monostearate,polyoxyethylene (20 OE) sorbitan tristearate, polyoxyethylene (8 OE)monostearate, hexaglyceryl monostearate, polyoxyethylene (10 OE)monostearate, polyoxyethylene (12 OE) distearate and polyoxyethylene (20OE) methylglucose distearate.

The cationic surfactants can be chosen from among the compounds citedhereinafter.

Nanoemulsions

The cationic particles can also be chosen from among the oil-in-waternanoemulsions containing an oily phase dispersed in an aqueous phase,wherein the oil globules have a number-average size less than 100 nm,characterized in that they comprise at least one amphiphilic lipidcomprising at least one nonionic amphiphilic lipid and one cationicamphiphilic lipid, the oily phase and the amphiphilic lipids beingpresent in a content such that the weight ratio of the oily phase toamphiphilic lipid ranges from 3 to 10.

The nanoemulsions generally have a bluish transparent appearance. Theirtransparency is measured by the coefficient of transmittance at 600nm—ranging from 10 to 90%- or else by turbidity. The turbidity of thecompositions of the invention ranges from 60 to 400 NTU and preferablyfrom 70 to 300 NTU, which turbidity is measured with the portable HACHturbidimeter, model 2100 P, at approximately 25° C.

The oil globules of the nanoemulsions of the invention have anumber-average size of less than 100 nm, preferably ranging from 20 to80 nm and more preferentially from 40 to 60 nm. Reduction of the size ofthe globules makes it possible to favor penetration of the active agentsinto the superficial layers of the skin (vehicle effect).

The nanoemulsions according to the invention are preferably prepared attemperatures ranging from 4 to 45° C., and so they are compatible withheat-sensitive active agents.

These dispersions are described in the following Applications inparticular: EP 0728460, EP 0879589, EP 1010413, EP 1010414, EP 1010416,EP 1013338, EP 1016453, EP 1018363, EP 1025898 and EP 1120102. In all ofthese applications, it is specified that an ionic surfactant will beadded to the nonionic surfactant (or mixture) in order to improve thestability of the particles.

In the case of the present application, exclusively one or more cationicsurfactants will be used as the ionic surfactant.

The proportions indicated in the foregoing references are to be adoptedand, as an example of cationic surfactants, the list common to all ofthe particles will be used.

The nonionic surfactants, preferably soluble or dispersible in water,contain at least one hydrophobic sequence and at least one hydrophilicsequence.

The nonionic amphiphilic lipids of the invention are preferably chosenfrom among;

1/ siliconized surfactants,

2/ amphiphilic lipids that are liquid at temperatures lower than orequal to 45° C., chosen from among the esters of at least one polyol andat least one fatty acid containing at least one saturated orunsaturated, linear or branched and especially unsaturated or branchedC₈ to C₂₂ alkyl chain, the polyol being chosen in the group formed bypolyethylene glycol containing 1 to 60 ethylene oxide units, sorbitan,glycerol, which can contain from 2 to 30 ethylene oxide units, and thepolyglycerols containing from 2 to 15 glycerol units.

3/ esters of fatty acid and sugar and ethers of fatty alcohol and sugar,

4/ surfactants that are solid at a temperature equal to 45° C., chosenfrom among the fatty esters of glycerol, the fatty esters of sorbitanand the fatty esters of oxyethylene sorbitan, the ethoxylated fattyethers and the ethoxylated fatty esters,

5/ block copolymers of ethylene oxide (A) and propylene oxide (B), andmixtures of these surfactants.

6/ The siliconized surfactants that can be used according to theinvention are siliconized compounds containing at least one oxyethylenechain —OCH₂CH₂— and/or oxypropylene chain —OCH₂CH₂CH₂—. There can becited those described in U.S. Pat. Nos. 5,364,633 and 5,411,744.

The siliconized surfactant used according to the present invention ispreferably a compound of formula (II):

in which:R₁, R₂, R₃, each independently of the others, represents a C₁ to C₆alkyl radical or a —(CH₂)_(x)—(OCH₂CH₂)_(y)—(OCH₂CH₂CH₂)_(z)—OR₄radical, wherein at least one radical R₁, R₂ or R₃ is not an alkylradical; R₄ being a hydrogen atom, an alkyl radical or an acyl radical;A is an integral number ranging from 0 to 200;B is an integral number ranging from 0 to 50; with the proviso that Aand B are not equal to zero at the same timex is an integral number ranging from 1 to 6;y is an integral number ranging from 1 to 30;z is an integral number ranging from 1 to 5.

According to a preferred embodiment of the invention, in the compound offormula (X), the alkyl radical is a methyl radical, x is an integralnumber ranging from 2 to 6 and y is an integral number ranging from 4 to30.

As an example of siliconized surfactants of formula (II), there can becited the compounds of formula (III):

in which A is an integral number ranging from 20 to 105, B is anintegral number ranging from 2 to 10 and y is an integral number rangingfrom 10 to 20.

As an example of siliconized surfactants of formula (II), there can alsobe cited the compounds of formula (IV):H—(OCH₂CH₂)_(y)—(CH₂)₃—[(CH₃)₂SiO]_(A′)—(CH₂)₃—(OCH₂CH₂)_(y)—OH  (IV)

in which A′ and y are integral numbers ranging from 10 to 20.

As siliconized surfactants there can be used in particular those sold bythe Dow Corning Co. under the names DC 5329, DC 7439-146, DC 2-5695 andQ4-3667. The compounds DC 5329, DC 7439-146, and DC 2-5695 are compoundsof formula (XI), in which, respectively, A is 22, B is 2 and y is 12; Ais 103, B is 10 and y is 12; A is 27, B is 3 and y is 12.

The compound Q4-3667 is a compound of formula (IV), in which A is 15 andy is 13.

2/ The amphiphilic lipids that are liquid at temperatures lower than orequal to 45° C. can be chosen in particular from among:

-   -   polyethylene glycol isostearate of molecular weight 400 (CTFA        name: PEG-8 isostearate), sold under the name Prisorine 3644 by        the UNICHEMA Co.;    -   diglyceryl isostearate, sold by the SOLVAY Co.;    -   polyglycerol laurate containing 2 glycerol units (polyglyceryl-2        laurate), sold under the name Diglycerin monolaurate by the        SOLVAY Co.;    -   sorbitan oleate, sold under the name SPAN 80 by the ICI Co.;    -   sorbitan isostearate, sold under the name NIKKOL SI 1 OR by the        NIKKO Co.;    -   α-butyl glucoside cocoate or α-butyl glucoside caprate, sold by        the ULICE Co.

3/ The esters of fatty acid and sugar that can be used as nonionicamphiphilic lipids in the nanoemulsion according to the invention arepreferably solid at a temperature lower than or equal to 45° C. and canbe chosen in particular in the group comprising the esters or themixtures of esters of C₈ to C₂₂ fatty acids and sucrose, maltose,glucose or fructose, and the esters or the mixtures of esters of C₁₄ toC₂₂ fatty acids and methylglucose.

The C₈ to C₂₂ or C₁₄ to C₂₂ fatty acids forming the fatty moiety of theesters that can be used in the nanoemulsion of the invention contain asaturated or unsaturated linear alkyl chain having respectively 8 to 22or 14 to 22 carbon atoms. The fatty moiety of the esters can be chosenin particular from among the stearates, behenates, arachidonates,palmitates, myristates, iaurates, caprates and mixtures thereof.Preferably stearates are used.

As examples of esters or mixtures of esters of fatty acids and sucrose,maltose, glucose or fructose there can be cited sucrose monostearate,sucrose distearate, sucrose tristearate and mixtures thereof, such asthe products sold by the Croda Co. under the names Crodesta F50, F70,F110 and F160, which respectively have an HLB (hydrophilic-lipophilicbalance) of 5, 7, 11 and 16; and as examples of esters or mixtures ofesters of fatty acids and methylglucose there can be cited thedistearate of methylglucose and polyglycerol-3, sold by the GoldschmidtCo. under the name Tego-care 450. There can also be cited the monoestersof glucose or maltose, such as methyl O-hexadecanoyl-6-D-glucoside andO-hexadecanoyl-6-D-maltoside.

The ethers of fatty alcohol and sugar that can be used as nonionicamphiphilic lipids in the nanoemulsion according to the invention aresolid at a temperature lower than or equal to 45° C. and can be chosenin particular in the group comprising the ethers or mixtures of ethersof C₈ to C₂₂ fatty alcohols and glucose, maltose, sucrose or fructose,and the ethers or the mixtures of ethers of C₁₄ to C₂₂ fatty alcoholsand methylglucose. In particular, they are alkyl polyglucosides.

The C₈ to C₂₂ or C₁₄ to C₂₂ fatty alcohols forming the fatty moiety ofthe ethers that can be used in the nanoemulsion of the invention containa saturated or unsaturated linear alkyl chain having respectively 8 to22 or 14 to 22 carbon atoms. The fatty moiety of the ethers can bechosen in particular from among the decyl, cetyl, behenyl, arachidyl,stearyl, palmityl, myristyl, lauryl, capryl and hexadecanoyl moietiesand mixtures thereof, such as cetearyl.

As examples of ethers of fatty alcohol and sugar there can be cited thealkyl polyglucosides such as decyl glucoside and lauryl glucoside sold,for example, by the Henkel Co. under the respective names of Plantaren2000 and Plantaren 1200, cetostearyl glucoside, possibly mixed withcetostearyl alcohol sold, for example, under the name Montanov 68 by theSeppic Co., under the name Tego-care CG90 by the Goldschmidt Co. andunder the name Emulgade KE3302 by the Henkel Co., as well as arachidylglucoside, for example in the form of the mixture of arachidic andbehenic alcohols with arachidyl glucoside, sold under the name Montanov202 by the Seppic Co.

As nonionic amphiphilic lipid of this type there are used moreparticularly sucrose monostearate, sucrose distearate, sucrosetristearate and mixtures thereof, the distearate of methylglucose andpolyglycerol-3. and the alkyl polyglucosides.

4/ The fatty esters of glycerol that can be used as nonionic amphiphiliclipids in the nanoemulsion according to the invention, solid at atemperature lower than or equal to 45° C., can be chosen in particularin the group comprising the esters formed from at least one acidcontaining a saturated linear alkyl chain having 16 to 22 carbon atomsand 1 to 10 glycerol moieties. One or more of these fatty esters ofglycerol can be used in the nanoemulsion of the invention.

These esters can be chosen in particular from among the stearates,behenates, arachidates, palmitates and mixtures thereof. Preferablystearates and palmitates are used.

As examples of the surfactant that can be used in the nanoemulsion ofthe invention there can be cited the monostearate, distearate,tristearate and pentastearate of decaglycerol (10 glycerol units) (CTFAnames: polyglyceryl-10 stearate, polyglyceryl-10 distearate,polyglyceryl-10 tristearate, polyglyceryl-10 pentastearate), such as theproducts sold under the respective names of Nikkol Decaglyn 1-S, 2-S,3-S and 5-S by the Nikko Co., and diglycerol monostearate (CTFA name:polyglyceryl-2 stearate), such as the product sold by the Nikko Co.under the name Nikkol DGMS.

The fatty esters of sorbitan that can be used as nonionic amphiphiliclipids in the nanoemulsion according to the invention, solid at atemperature lower than or equal to 45° C., can be chosen in particularin the group comprising the esters of C₁₆ to C₂₂ fatty acids andsorbitan and the esters of C₁₆ to C₂₂ fatty acids and oxyethylenesorbitan. They are formed from at least one fatty acid containing atleast one saturated linear alkyl chain having respectively 16 to 22carbon atoms, and from sorbitol or ethoxylated sorbitol. The oxyethyleneesters generally contain 1 to 100 ethylene oxide units and preferably 2to 40 ethylene oxide units (OE).

These esters can be chosen in particular from among the stearates,behenates, arachidates, palmitates and mixtures thereof. Preferablystearates and palmitates are used.

As examples of fatty esters of sorbitan and fatty esters of oxyethylenesorbitan that can be used in the nanoemulsion of the invention there canbe cited sorbitan monostearate (CTFA name: sorbitan stearate), sold bythe ICI Co. under the name Span 60, sorbitan monopalmitate (CTFA name:sorbitan palmitate), sold by the ICI Co. under the name Span 40,sorbitan (20 OE) tristearate (CTFA name: polysorbate 65), sold by theICI Co. under the name Tween 65.

The ethoxylated fatty ethers that are solid at a temperature lower thanor equal to 45° C. that can be used as nonionic amphiphilic lipids inthe nanoemulsion according to the invention are preferably ethers formedfrom 1 to 100 ethylene oxide units and at least one fatty alcohol chainhaving 16 to 22 carbon atoms. The fatty chain of the ethers can bechosen in particular from among the behenyl, arachidyl, stearyl andcetyl moieties and mixtures thereof, such as cetearyl. As examples ofethoxylated fatty ethers there can be cited the ethers of behenicalcohol containing 5, 10, 20 and 30 ethylene oxide units (CTFA names:beheneth-5, beheneth-10, beheneth-20, beheneth-30), such as the productssold under the names Nikkol BB5, BB10, BB20 and BB30 by the Nikko Co.,and stearyl alcohol ether containing 2 ethylene oxide units (CTFA name:steareth-2), such as the product sold under the name Brij 72 by the ICICo.

The ethoxylated fatty esters that are solid at a temperature lower thanor equal to 45° C. that can be used as nonionic amphiphilic lipids inthe nanoemulsion according to the invention are esters formed from 1 to100 ethylene oxide units and at least one fatty acid chain having 16 to22 carbon atoms. The fatty chain of the esters can be chosen inparticular from among the stearate, behenate, arachidate and palmitatemoieties and mixtures thereof. As examples of ethoxylated fatty estersthere can be cited the stearic acid ester containing 40 ethylene oxideunits, such as the product sold under the name My j 52 by the ICI Co.(CTFA name: PEG-40 stearate), as well as the ester of behenic acidcontaining 8 ethylene oxide units (CTFA name: PEG-8 behenate), such asthe product sold under the name Compritol HD5 ATO by the Gattefosse Co.

5/ The block copolymers of ethylene oxide and propylene oxide that canbe used as nonionic amphiphilic lipids in the nanoemulsion according tothe invention can be chosen in particular from among the blockcopolymers of formula (V):—HO(C₂H₄O)x(C₃H₆O)y(C₂H₄O)zH  (V)

in which x, y and z are integral numbers such that x+z ranges from 2 to100 and y ranges from 14 to 60, and mixtures thereof, and moreparticularly from among the block copolymers of formula (V) having anHLB ranging from 2 to 16.

These block copolymers can be chosen in particular from among thepoloxamers, and in particular from among poloxamer 231, such as theproduct sold by the ICI Co. under the name Pluronic L81 of formula (V),with x=z=6, y=39 (HLB 2); poloxamer 282, such as the product sold by theICI Co. under the name Pluronic L92 of formula (V), with x=z=10, y=47(HLB 6); and poloxamer 124, such as the product sold by the ICI Co.under the name Pluronic L44 of formula (V), with x=z=11, y=21 (HLB 16).

As nonionic amphiphilic lipids there can also be cited the mixtures ofnonionic surfactants described in European Patent A 705593, incorporatedhere by reference.

Among the nonionic amphiphilic lipids there can be used in particular:

-   -   PEG 400 isostearate or PEG-8 isostearate (containing 8 moles of        ethylene oxide),    -   diglyceryl isostearate,    -   polyglycerol monolaurate containing 2 glycerol units and        polyglycerol stearates containing 10 glycerol units.    -   sorbitan oleate,    -   sorbitan isostearate and mixtures thereof.

The nonionic amphiphilic lipids can be present in the nanoemulsionaccording to the invention in a content ranging from 0.2% to 12% byweight relative to the total weight of the composition, and preferablyranging from 0.2% to 8% by weight, and preferentially ranging from 0.2%to 6% by weight.

The cationic amphiphilic lipids are chosen from among the list givenhereinafter.

They are present in the nanoemulsions of the invention in concentrationsranging preferably from 0.01 to 6% by weight relative to the totalweight of the nanoemulsion, and more particularly from 0.2 to 4% byweight.

Oils:

The oily phase of the nanoemulsion according to the invention comprisesat least one oil. The oils that can be used in the nanoemulsions of theinvention are preferentially chosen in the group formed by:

-   -   the oils of animal or vegetable origin, formed by esters of        fatty acids and polyols, particularly the liquid triglycerides,        such as sunflower, corn, soy, avocado, jojoba, pumpkin,        grapeseed, sesame and hazelnut oils, fish oils, glycerol        tricaprocaprylate, or the vegetable or animal oils of formula        R₉COOR₁₀, in which R₉ represents the residue of a higher fatty        acid containing 7 to 29 carbon atoms and R₁₀ represents a linear        or branched hydrocarbon chain containing 3 to 30 carbon atoms,        particularly alkyl or alkenyl, examples being purcellin oil or        liquid jojoba wax;    -   natural or synthetic essential oils such as, for example,        eucalyptus, lavandin, lavender, vetiver, litsea cubeba, citron,        sandalwood, rosemary, chamomile, savory, nutmeg, cinnamon,        hyssop, caraway, orange, geraniol, cade and bergamot oil;    -   synthetic oils such as parleam oil, polyolefins and liquid        carboxylic acid esters;    -   mineral oils such as hexadecane, isohexadecane and paraffin oil;    -   halogenated oils, especially fluorocarbons such as fluoro        amines, for example perfluorotributylamine, fluorinated        hydrocarbons, for example perfluorodecahydronaphthalene, fluoro        esters and fluoro ethers;    -   volatile or non-volatile silicone oils.

The polyolefins that can be used as synthetic oils are in particular thepoly-α-olefins and more particularly those of the hydrogenated ornon-hydrogenated polybutene type, and preferably hydrogenated ornon-hydrogenated polyisobutene.

The liquid carboxylic acid esters that can be used as synthetic oils canbe esters of mono-, di-, tri- or tetracarboxylic acids. The total carbonnumber of the esters is generally greater than or equal to 10 andpreferably less than 100, and more particularly less than 80. Suchesters are in particular the monoesters of saturated or unsaturated,linear or branched C₁ to C₂₆ aliphatic acids and saturated orunsaturated, linear or branched C₁ to C₂₆ aliphatic alcohols, the totalcarbon number of the esters generally being greater than or equal to 10.It is also possible to use the esters of C₄ to C₂₂ di- or tricarboxylicacids and C₁ to C₂₂ alcohols and the esters of C₂ to C₂₆ mono-, di- ortricarboxylic acids and di-, tri-, tetra or pentahydroxy alcohols.

Among the esters cited in the foregoing, there are preferably used alkylpalmitates such as ethyl palmitate, isopropyl palmitate, ethyl-2-hexylpalmitate and 2-octyldecyl palmitate; alkyl myristates such as isopropylmyristate, butyl myristate, cetyl myristate and 2-octyldodecylmyristate; alkyl stearates, such as hexyl stearate, butyl stearate andisobutyl stearate; alkyl malates, such as dioctyl malate; alkyllaurates, such as hexyl laurate and 2-hexyldecyl laurate; isononylisononanate; cetyl octanoate.

Advantageously, the nanoemulsion according to the invention contains atleast one oil of molecular weight greater than or equal to 400,especially ranging from 400 to 10,000, better ranging from 400 to 5000,or even ranging from 400 to 5000. The oils of molecular weight greaterthan or equal to 400 can be chosen from among the oils of animal orvegetable origin, the mineral oils, the synthetic oils and the siliconeoils and mixtures thereof. As oils of this type there can be cited, forexample, isocetyl palmitate, isocetyl stearate, avocado oil and jojobaoil.

The nanoemulsions according to the invention contain a quantity of oilyphase (oil and other fatty substances not including the amphiphilicliquid) ranging preferably from 2 to 40% by weight relative to the totalweight of the nanoemulsion, and more particularly from 4 to 30% byweight and preferentially from 4 to 20% by weight.

The oily phase and the amphiphilic lipids (nonionic and ionicamphiphiles) are preferably present in the nanoemulsion according to theinvention in a weight ratio of the quantity of oily phase to thequantity of amphiphilic liquid that ranges from 3 to 10 andpreferentially from 3 to 6. By “quantity of oily phase” there isunderstood here the total quantity of the constituents of this oilyphase without including the quantity of amphiphilic liquids.

Besides the urea derivatives of formula (I) described previously, thenanoemulsions according to the present invention can contain solvents,in particular to improve the transparency of the composition ifnecessary.

These solvents are preferably chosen in the group formed by:

-   -   C₁ to C₈ lower alcohols, such as ethanol;    -   glycols, such as glycerol, propylene glycol, 1,3-butylene        glycol, dipropylene glycol and the polyethylene glycols        containing 4 to 16 and preferably 8 to 12 ethylene oxide units;    -   sugars, such as glucose, fructose, maltose, lactose and sucrose.

These solvents can be used as mixtures. When they are present in thenanoemulsion of the invention, they can be used in concentrationsranging preferably from 0.01 to 30% by weight relative to the totalweight of the nanoemulsion, and better from 5 to 20% by weight relativeto the total weight of the nanoemulsion. The quantity of alcohol(s)and/or of sugar(s) preferably ranges from 5 to 20% by weight relative tothe total weight of the nanoemulsion and the quantity of glycolspreferably ranges from 5 to 15% by weight relative to the total weightof the nanoemulsion.

Preparation Method:

The method for preparation of a nanoemulsion such as defined in theforegoing comprises mixing the aqueous phase containing the ureaderivative with the oily phase under vigorous agitation at a temperatureranging from 10° C. to 80° C., performing a step of high-pressurehomogenization at a pressure above 5·10⁷ Pa and if necessary adding thepolymer used. According to a preferred embodiment of the invention,another step of high-pressure homogenization at a pressure above 5·10⁷Pa is then performed. The high-pressure homogenization is preferablyperformed at a pressure ranging from 6·10⁷ to 18·10⁷ Pa. The shearpreferably ranges from 2·10⁶ s⁻¹ to 5·10⁸ s⁻¹ and better from 1·10⁸ s⁻¹to 3·10⁸ s⁻¹ (s⁻¹ denotes seconds⁻¹). Such a method makes it possible toobtain nanoemulsions that are compatible with heat-sensitive activecompounds and that can contain oils, and especially perfumes containingfatty substances, without denaturing them.

Nanocapsules

The nanocapsules useful in the invention are those described in PatentApplications EP 0447318, EP 0557489, EP 0780115, EP 1025901, EP 1029587,EP 1034839, EP 1414390, FR 2830776, EP 1342471, FR 2848879 and FR04/50057.

Nanocapsules are core-shell particles having an oily core and a polymershell. The different applications cited in the foregoing relate todifferent polymer families and different methods for obtaining same. Thesize of the capsules is always less than 1 μm, and it is possible toobtain sizes less than 80 nm. These particles can be coated by alamellar liquid crystal phase, most often composed of a lecithin or of adimethicone copolyol. The coating must be composed of an amphiphiliclipid capable of spontaneously forming a lamellar liquid crystal phaseupon contact with water. It is to this amphiphilic lipid capable offorming a lamellar phase that there will be added the cationicsurfactant that will impart a positive zeta potential to the particles(the nanocapsule). The weight ratio between the amphiphilic lipidforming the lamellar phase and the cationic surfactant will rangebetween 99/1 and 75/25.

The cationic surfactants that can be used are those listed hereinafter.

Organic Particles

The organic particles of the invention are solid nanospheres withoutinternal cavity, formed by different methods (dispersion in water,nanoprecipitation, microemulsion, etc.) and are composed of at least onepolymer or of at least one copolymer or of a mixture thereof. Theparticle is cationic, with the zeta potential defined in the foregoing,either because the polymer or copolymer or the polymers or copolymersare cationic, or because they are nonionic and a cationic surfactantsuch as described hereinafter is used. Relative to the polymer, theproportion of cationic surfactant will range between 0 and 25%.

Inorganic Particles

The cationic inorganic particles of the invention can be based, forexample, on silica, TiO₂, ZnO, alumina, etc. As an example, there willbe cited alumina particles in colloidal dispersion in water, such asNanomer 2 of Nalco. Clariant and Grace also propose particles of thistype.

Cationic Surfactants that can be Used for Preparation of the CationicParticles of the Invention

The cationic surfactants that can be used according to the invention arelisted hereinafter. This list is non-limitative.

The cationic amphiphilic lipids are preferably chosen in the groupformed by the quaternary ammonium salts, the fatty amines and the saltsthereof.

The quaternary ammonium salts are, for example:

-   -   those having the following general formula (IV):

in which the radicals R1 to R4, which can be identical or different,represent a linear or branched aliphatic radical containing 1 to 30carbon atoms, or an aromatic radical such as aryl or alkylaryl. Thealiphatic radicals can contain hetero atoms, such as, in particular,oxygen, nitrogen, sulfur and halogens. The aliphatic radicals arechosen, for example, from among the alkyl, alkoxy, polyoxyalkylene(C2 toC6), alkylamide, alkyl(C12 to C22)amidoalkyl(C2 to C6), alkyl(C12 toC22) acetate and hydroxyalkyl containing approximately 1 to 30 carbonatoms; X is an anion chosen in the group of halides, phosphates,acetates, lactates, alkyl(C2 to C6) sulfates, alkyl- oralkylarylsulfonates,

-   -   the quaternary ammonium salts of imidazolinium, such as, for        example, that of the following formula (V):        in which R5 represents an alkenyl or alkyl radical that contains        8 to 30 carbon atoms and is derived, for example, from tallow        fatty acids, R6 represents a hydrogen atom, a C1 to C4 alkyl        radical or an alkenyl or alkyl radical containing 8 to 30 carbon        atoms, R7 represents a C1 to C4 alkyl radical, R8 represents a        hydrogen atom or a C1 to C4 alkyl radical, X is an anion chosen        in the group of halides, phosphates, acetates, lactates, alkyl        sulfates and alkyl- or alkylarylsulfonates. Preferably, R5 and        R6 denote a mixture of alkenyl or alkyl radicals that contain 12        to 21 carbon atoms and are derived, for example, from tallow        fatty acids, R7 denotes methyl and R8 denotes hydrogen. Such a        product is sold, for example, under the name “REWOQUAT W 75” by        the REWO Co.,

the quaternary diammonium salts of formula (VI):

in which R9 denotes an aliphatic radical containing approximately 16 to30 carbon atoms, R10, R11, R12, R13 and R14, identical or different, arechosen from among hydrogen or an alkyl radical containing 1 to 4 carbonatoms, and X is an anion chosen in the group of halides, acetates,phosphates, nitrates and methyl sulfates. Such quaternary diammoniumsalts comprise in particular propane tallow diammonium dichloride;

the quaternary ammonium salts containing at least one ester function

The quaternary ammonium salts that contain at least one ester functionand that are usable according to the invention are, for example, thoseof the following formula (VII):

in which:

-   -   R15 is chosen from among the C1 to C6 alkyl radicals and the C1        to C6 hydroxyalkyl or dihydroxyalkyl radicals;    -   R16 is chosen from among:    -   the radical    -   linear or branched, saturated or unsaturated C1 to C22        hydrocarbon radicals R20,    -   a hydrogen atom,    -   R18 is chosen from among:    -   the radical    -   linear or branched, saturated or unsaturated C1 to C6        hydrocarbon radicals R22,    -   a hydrogen atom,    -   R17, R19 and R21, identical or different, are chosen from among        the linear or branched, saturated or unsaturated C7 to C21        hydrocarbon radicals:    -   n, p and r, identical or different, are integers equal to 2 to        6;    -   y is an integer equal to 1 to 10;    -   x and z, identical or different, are integers equal to 0 to 10;    -   X— is a simple or complex, organic or inorganic anion; with the        provisos that the sum of x+y+z is equal to 1 to 15, that when x        is equal to 0 then R16 denotes R20 and that when z is equal to 0        then R18 denotes R22.

The alkyl radicals R15 can be linear or branched and more particularlylinear.

Preferably R15 denotes a methyl, ethyl, hydroxyethyl or dihydroxypropylradical, and more particularly a methyl or ethyl radical.Advantageously, the sum of x+y+z is equal to 1 to 10.

When R16 is a hydrocarbon radical R20, it can be long with 12 to 22carbon atoms or short with 1 to 3 carbon atoms.

When R18 is a hydrocarbon radical R22, it preferably has 1 to 3 carbonatoms.

Advantageously, R17, R19 and R21, identical or different, are chosenfrom among the linear or branched, saturated or unsaturated C11 to C21hydrocarbon radicals, and more particularly from among the linear orbranched, saturated or unsaturated C11 to C21 alkyl and alkenylradicals.

Preferably x and z, identical or different, are equal to 0 or 1.

Advantageously, y is equal to 1.

Preferably, n, p and r, identical or different, are equal to 2 or 3 andeven more particularly are equal to 2.

The anion is preferably a halide (chloride, bromide or iodide) or analkyl sulfate, more particularly methyl sulfate. However, it is possibleto use methanesulfonate, phosphate, nitrate, tosylate, an anion derivedfrom an organic acid such as acetate or lactate, or any other anion thatis compatible with ammonium and has an ester function.

The anion X— is even more particularly chloride or methyl sulfate.

More particularly, there are used the ammonium salts of formula (VII) inwhich:

R15 denotes a methyl or ethyl radical,

x and y are equal to 1;

z is equal to 0 or 1;

n, p and r are equal to 2;

R16 is chosen from among:

the radical

the methyl, ethyl or C14 to C22 hydrocarbon radicals

a hydrogen atom;

R18 is chosen from among:

the radical

a hydrogen atom;

R17, R19 and R21, identical or different, are chosen from among thelinear or branched, saturated or unsaturated C13 to C17 hydrocarbonradicals, and preferably from among the linear or branched, saturated orunsaturated C13 to C17 alkyl and alkenyl radicals.

Advantageously, the hydrocarbon radicals are linear.

As examples there can be cited the compounds of formula (VII) such asthe salts (especially chloride or methyl sulfate) ofdiacyloxyethyldimethylammonium, ofdiacyloxyethylhydroxyethylmethylammoniurn, ofmonoacyloxyethyldihydroxyethylmethylammonium, oftriacyloxyethylmethylammonium, ofmonoacyloxyethylhydroxyethyldimethylammonium and mixtures thereof. Theacyl radicals preferably have 14 to 18 carbon atoms and originate moreparticularly from a vegetable oil such as palm or sunflower oil.

When the compound contains a plurality of acyl radicals, these radicalsmay be identical or different.

These products are obtained, for example, by direct esterification oftriethanolamine, triisopropanolamine, alkyldiethanolamine oralkyldiisopropanolamine, which may be oxyalkenated on the fatty acids oron mixtures of fatty acids of vegetable or animal origin or bytransesterification of their methyl esters. This esterification isfollowed by quaternization by means of an alkylating agent such as analkyl halide (preferably methyl or ethyl), a dialkyl sulfate (preferablymethyl or ethyl), methyl methylsulfonate, methyl paratoluenesulfonate,or glycol or glycerol chlorohydrin.

Such compounds are sold, for example, under the names DEHYQUART by theHENKEL Co., STEPANQUAT by the STEPAN Co., NOXAMIUM by the CECA Co. andREWOQUAT WE 18 by the REWOWITCO Co.

The composition according to the invention preferably contains a mixtureof salts of mono-, di- and triesters of quaternary ammonium, wherein thediester salts represent the majority by weight.

As the mixture of ammonium salts there can be used, for example, themixture containing 15 to 30% by weight ofacyloxyethyldihydroxyethylmethylammonium methyl sulfate, 45 to 60% ofdiacyloxyethylhydroxyethylmethylammonium methyl sulfate and 15 to 30% oftriacyloxyethylmethylammonium methyl sulfate, the acyl radicals having14 to 18 carbon atoms and originating from palm oil, which may be partlyhydrogenated. There can also be used the ammonium salts containing atleast one ester function described in U.S. Pat. No. 4,874,554 and4,137,180.

Among the quaternary ammonium salts of formula (IV) there are preferred,on the one hand, the tetraalkylammonium chlorides such as, for example,the dialkyldimethylammonium or alkyltrimethylammonium chlorides, inwhich the alkyl radical contains approximately 12 to 22 carbon atoms,especially behenyltrimethylammonium, distearyldimethylammonium,cetyltrimethylammonium and benzyldimethylstearylammonium chlorides, orelse, on the other hand, stearamidopropyldimethyl(myristylacetate)ammonium chloride, sold under the name “CERAPHYL 70” by the VANDYK Co.

According to the invention, behenyltrimethylammonium chloride or bromideand CTAB (cetyltrimethylammonium bromine) are the most particularlypreferred quaternary ammonium salts.

The fatty amines of the invention correspond to the general formula:

-   -   R₁ is a saturated or unsaturated, branched or linear hydrocarbon        chain having between 8 and 30 and preferably between 10 and 24        carbon atoms.    -   R₂ and R₃ are selected independently from among saturated or        unsaturated, branched or linear hydrocarbons having between 1        and 10 and preferably between 1 and 4 carbon atoms.    -   R₂ and R₃ can also correspond to a hydrogen atom H, again        independently of one another.    -   M is between 1 and 10 and preferably between 1 and 5.

As non-limitative examples there will be cited: stearylamine, stearateaminoethylethanolamide, stearyl diethanolamide, stearatediethylenetriamine, stearamidopropyldimethylamine,stearamidopropyldiethylamine, stearamidoethyldiethylamine,stearamidoethyldimethylamine, palmitamidopropyldimethylamine,palmitamidopropyldiethylamine, palmitamidoethyldiethylamine,palmitamidoethyldimethylamine, behenamidopropyldimethylamine,behenamidopropyldiethylmine, behenamidoethyldiethylamine,behenamidoethyldimethylamine, arachidamidopropyldimethylamine,arachidamidopropyldiethylamine, arachidamidoethyldiethylamine,arachidamidoethyldimethylamine.

As commercially available fatty amine there can be cited Incromine BB ofCroda, Amidoamine MSP of Nikkol and the Lexamines of Inolex.

As other fatty amines there can be cited, as examples, stearylamine,stearate aminoethylethanolamide, stearyl diethanolamide and stearatediethylenetriamine, which Sabo sells among others with the Sabominaseries.

There will also be cited the fatty amine acetates, such as the Acetamineseries of Kao Corp.

These fatty amines can also be ethoxylated, such as Berols 380, 390, 453and 455, the Ethomeens of Akzo Nobel or Marlazins L10, OL2, OL20, T15/2and T50 of Condea Chemie.

The particles of the present invention can be introduced into allmedicinal supports intended for cosmetic, dermatological and ophthalmicpurposes. As examples, there will be cited lotions, serums, gels and alltypes of emulsions.

The double stranded RNA oligonucleotides (or else dsRNA for doublestranded RNA, siRNA for short interfering RNA) that can be usedaccording to the present invention are double stranded nucleic acidfragments capable of totally or partly inhibiting gene expression in aeukaryotic cell, these double stranded RNA oligonucleotides generallycontaining between 12 and 40 nucleotides, preferentially from 20 to 25nucleotides. These double stranded oligonucleotides are composed of onesense strand and one anti-sense strand, corresponding to the sequence ofthe target messenger RNA to be degraded. The double stranded RNAoligonucleotide has free or unpaired ends of 2 to 6 nucleotides.

It will be possible for the double stranded RNA oligonucleotidesaccording to the invention to be double stranded RNA oligonucleotidescontaining one or more nucleotides modified by substitution, deletion orinsertion, and these modifications will be such that the oligonucleotidesequence of the double stranded RNA will permit it to recognizespecifically a fragment of the target mRNA of the degradation mechanism.

It will also be possible for the oligonucleotides of the double strandedRNA to have a modified skeleton that, for example, imparts betterstability to it.

For example, the phosphodiester bonds of natural RNA strands can bemodified to include at least one nitrogen or sulfur atom. In addition,the double stranded RNA oligonucleotides according to the invention cancomprise bases other than the 4 classical bases.

The double stranded structure of the double stranded RNA oligonucleotidecan be obtained by pairing two complementary single RNA strands or by aunique “self-complementary” single RNA strand, or in other words onecomprising two fragments of complementary sequences that can be pairedby folding the single strand to form a double helix.

Examples of double stranded RNA oligonucleotides are described inInternational Patent Applications WO 00/44895, WO 01/36646, WO 99/32619,WO 01/29058, WO 00/44914 or else WO 03/101376.

They may also be oligonucleotides of the double stranded RNA known as“stealth RNA”, such as those sold by the Invitrogen Co. and described inUS Patent Applications 2004014956 and 2004054155, which areoligonucleotides of double stranded RNA that can be modified in such away that, as a non-limitative example, one of its ends contains a2′-O-methyl group.

The double stranded RNA oligonucleotides can be synthesized manually orautomatically by numerous in vivo or in vitro synthesis methods as knownin the art.

The in vitro synthesis methods can be chemical or enzymatic, for exampleby using a polymerase RNA (such as T3, T7 or SP6, for example), whichwill achieve transcription of a chosen DNA (or cDNA) sequence model.

Numerous methods for in vivo synthesis of double stranded RNA aredescribed in the literature. They can be achieved in various cellulartypes of bacteria or higher organisms (Sambrook et al. MolecularCloning, A Laboratory Manual, Second Edition (1989), DNA cloning, volumeI and II, D. N. Glover (ed. 1985), Oliginucleotide Synthesis, M. J.Gaits (ed. 1984), Nucleic Acid Hybridation, B. D. Hames and S. J.Higgins (ed. 1984), Transcription and Translation B. D. Hames and S. J.Higgins (ed. 1984), Animal Cell Culture, R. I. Freshney (ed. 1986),Immobilised Cells and Enzymes, IRL Press (1986), B. Pertal, A PracticalGuide to Molecular Cloning (1984), Gene Transfer Vectors for MammalianCells, J. H. Miller and M. P. Calos, Cold Spring Harbor Laboratory (ed.1987), Methods in Enzymology, vol. 154, Wu and Grossman, and 155, Wu,Mayer and Walker (1987), Immunochemical Methods in Cell and MolecularBiology, Academic Press, London, Scopes (1987), Protein Purification:Principle and Practice, 2^(nd) ed., Springer-Verlag, N.-Y. and Handbookof Experimental Immunology, vol. I-IV, C. D. Weir and C. C. Blackwell(1986)). Reference can also be made to the synthesis methods describedin Patent Applications WO01/36646, WO01/75164 and US20030088087.

The person skilled in the art will choose a double stranded RNAoligonucleotide concentration appropriate for the intended use and theactivity of the chosen oligonucleotide. It will be possible, withoutthis being limitative, for the double stranded RNA oligonucleotideconcentration to be between 1 nM and 100 mM.

The compositions can also contain one or more ingredients, such ascarriers, active agents, etc suitable for cosmetic and/or ocularapplications. Such ingredients are well-known in the art.

A second object of the present invention relates to the topical orocular use of a composition comprising at least one double stranded RNAoligonucleotide associated with at least one cationic particle of sizesmaller than or equal to 1 μm, preferably smaller than or equal to 500nm or 300 nm, of zeta potential between 10 and 80 mV, chosen from amongsurfactant micelles, block polymer micelles, liposomes of nonionic andcationic surfactants, niosomes, oleosomes, particles of nanoemulsions,nanocapsules, organic particles or inorganic particles for cosmeticpurposes.

Another object of the present invention relates to a cosmetic treatmentmethod comprising the topical or ocular application of a compositioncomprising at least one double stranded RNA oligonucleotide and at leastone cationic particle of size smallerless than or equal to 1 μm, of zetapotential between 10 and 80 mV, chosen from among surfactant micelles,block polymer micelles, liposomes of nonionic and cationic surfactants,niosomes, oleosomes, particles of nanoemulsions, nanocapsules, organicparticles or inorganic particles.

For purposes of cosmetic, dermatological, ophthalmic or pharmaceuticalcare, it is sought to avoid invasive methods such as subcutaneous orocular injections. The cationic particles associated with the siRNAaccording to the present invention can be applied topically on the skin,the mucous membranes or in the eye, in suspension or incorporated in anacceptable cosmocentric support, such as lotions, serum, emulsified ornon-emulsified gels, oil-in-water, water-in-oil and multiple emulsions,microemulsions, etc. Care will be taken that this introduction into acosmetic support does not jeopardize the stability of the particlesassociated with the siRNA.

As a non-limitative example, it will be possible to design thecomposition according to the invention, comprising dsRNA vectorized withcationic particles, to prevent and/or to treat actinic or chronologicalaging, wrinkles, loss of cutaneous firmness of the skin of the faceand/or of the body, seborrheic disorders, greasy skin, dry skin andpigment spots. These compositions can also be designed to slow hairloss, to stimulate hair growth, to improve hair quality, to limit orprevent regrowth of unwanted hair, etc.

These compositions can also be designed to treat cutaneous orophthalmologic disorders.

To favor penetration of the particles associated with the siRNA it willbe possible to modify the permeability of the skin; thus it is possibleto control the permeability of the skin by measuring the IWL (insensiblewater loss) with a Tewameter TM210 or with a Dermalab of CortexTechnology.

As an example, it will be possible to use the following methods:

-   -   stripping (corneodisk, “varnish”), chemical peeling or        mechanical dermabrasion;    -   pretreatment by a mixture of one or more solvents having a        delipidating effect;    -   cleaning of the skin by a detergent foaming product;    -   occlusive pre- or post-treatment, for example either by covering        the surface of the skin to be treated with an impermeable        synthetic membrane (such as Blenderm) or by application of a        coat of vaseline. This has the effect of blocking the natural        IWL of the skin and causing super-hydration of the epidermal        lipids, which thus become more permeable.

After application of the composition of the present invention, it willalso be possible to use methods known to the person skilled in the artfor favoring penetration of molecules into the skin, examples beingiontophoresis or electroporation.

Finally, the present invention has as an object the use of cationicparticles of size smaller than or equal to 1 μm, of zeta potentialbetween 10 and 80 mV, chosen from among surfactant micelles, blockpolymer micelles, liposomes of nonionic and cationic surfactants,niosomes, oleosomes, particles of nanoemulsions, nanocapsules, organicparticles or inorganic particles as agents that favor topical or ocularpenetration of at least one double stranded RNA oligonucleotide.

The following figures illustrate the tests described in Example 2:

I. Cell Targeting in Episkin—Day 6

Fig. I-1: Block-It Fluorescent Oligo (DAPI)

Fig. I-2: Block-It Fluorescent Oligo+Lipofectamine 2000 (DAPI)

Fig. I-3: Block-It Fluorescent Oligo+E2 (×40, DAPI)

Fig. I-4: E2 (×40, DAPI)

Fig. I-5: Block-It Fluorescent Oligo+E2 (×100, DAPI)

Fig. I-6: Block-It Fluorescent Oligo+E2 (×100)

II. Cell Targeting in Episkin—Day 13

Fig. II-1: Block-It Fluorescent Oligo (×40, DAPI)

Figs. IIIA and IIIB: visualization of the effect of the formulationcomprising octyl glucoside and CTAB in different concentrations on theviability of HaCaT cells.

The invention will now be illustrated by the following non-limitingexamples. In these examples, the amounts are indicated as percentages byweight.

EXAMPLE 1 Formulations

Cationic Micelles:

Octyl β-glucoside/cetyltriammonium bromide in the molar ratio of 5/1

E1: 100 mM in distilled water

E2: 50 mM in distilled water

E3: 25 mM in distilled water

E4: 12.5 mM in distilled water

The 2 surfactants (nonionic+cationic) are solubilized in distilledwater. A suspension of siRNA (20 μM) is added in a volume(siRNA:micelles) of between 1:1 and 1:9, thus proportionally reducingthe micellar concentration.

Micelles of decyl β-glucoside/behenyltriammonium chloride in the molarratio of 5/1 in distilled water. The same concentrations are prepared asin the preceding example.

Cationic Liposomes

EXAMPLE 1 “fluid” Vesicles

PEG 400 isostearate 5.5% Behenyltriammonium chloride 0.5% Distilledwater qsp 100

EXAMPLE 2 “Rigid” Vesicles

Sorbitan palmitate 2.75% Cholesterol 2.75% Behenyltriammonium chloride 0.5% Distilled water qsp 100

These examples of liposome suspensions are prepared by dialysis. Theconstituent lipids of the vesicles are solubilized in an aqueoussolution of octyl β-glucoside. This solution is then dialyzed againstwater for 72 h.

The suspension of siRNA is added, making the vesicle concentrationaround 3% of lipid, this value being merely indicative.

Cationic Oleosomes Oily phase Sucrose mono/di-stearate sold byStearainerie Dubois 0.45% Sorbitan (4 OE) stearate (Tween 61 Uniquema)0.30% Behenyltriammonium chloride 0.21% Vitamin E acetate  0.5% Jojobaoil  0.5% Stearyl heptanoate   1% Volatile silicone oil SE   1% VitaminF glyceride  0.5% Preservative 0.02% BHT 0.01% Aqueous phase Distilledwater qsp 100 Preservative  0.1%

This dispersion is prepared by high-pressure homogenization in order toobtain a particle size of approximately 170 nm. There is then added asuspension of siRNA (20 μM) in ratios ranging from 1:1 to 1:20(siRNA:oleosomes). The siRNA will then become complexed on the surfaceof the particles.

Cationic Nanocapsules Organic phase Polycaprolactone (MW: 50000)   1%Vitamin E   1% Dimethicone copolyol DC2 5695 (Dow Corning) 0.5%Behenyltriammonium chloride 0.21%  Acetone 200 ml Aqueous phase PluronicF68 0.5% Distilled water 200 ml

The organic phase is introduced into the aqueous phase with agitation.The acetone and 100 ml of the aqueous phase are then evaporated toobtain the suspension of nanocapsules, which have a size of 220 nm.There is then added a suspension of siRNA (20 μM) in ratios ranging from1:1 to 1:20 (siRNA:nanocapsules). The siRNA will then become complexedon the surface of the particles.

Organic Nanoparticles Organic phase Polyethylene adipate (ScientificPolymer Products)   2% Dimethicone copolyol DC2 5695 (Dow Corning) 0.5%Behenyltriammonium chloride 0.21%  Acetone 200 ml Aqueous phase PluronicF68 0.5% Distilled water 200 ml

The organic phase is introduced into the aqueous phase with agitation.The acetone and 100 ml of the aqueous phase are then evaporated toobtain the suspension of nanoparticles, which have a size of 180 nm.There is then added a suspension of siRNA (20 μM) in ratios ranging from1:11 to 1:20 (siRNA:nanoparticles). The siRNA will then become complexedon the surface of the particles.

Cationic Nanoemulsion Oily phase PEG 400 isostearate sold by Uniqema 1%Behenyltriammonium chloride 1% Avocado oil 1% Jojoba oil 3%Cyclopentamethylsiloxane 2% Aqueous phase Distilled water 30% Dipropylene glycol 10%  Dilution phase Distilled water qsp 100%Preservative 0.1%  

An emulsion is prepared by dispersing the oily phase in the aqueousphase under very vigorous agitation. The suspension obtained is thenhomogenized several times by means of a very high-pressure homogenizer,at a pressure of approximately 1200 b. The size of the particles is onthe order of 50 nm, and the suspension is transparent. The dilutionphase is then added.

As in the preceding cases, there is then added a suspension of siRNA (2μM) in ratios ranging from 1:1 to 1:20 (siRNA:nanoemulsion). The siRNAwill then become complexed on the surface of the particles.

EXAMPLE 2 Cell Targeting

Cell and tissue targeting was carried out in the reconstructed epidermismodel developed by EPISKIN SNC. Two phases of growth kinetics of themodel were chosen for topical application of the micelle/siRNA complex:

1 application on day 6 of growth of the epidermis, corresponding to anepidermis at the beginning of stratification and keratinization.

1 application on day 13 of growth of the epidermis, corresponding to astratified and keratinized epidermis.

The siRNA chosen is Block-It Fluorescent Oligo (20 μM), such asdescribed in the manual “Block-It Transfection Kit” (Catalog ref. No.:13750-070, Invitrogen). It is a 25-nucleotide double stranded RNAcoupled with fluorescein. The coded sequence has no homology with thehuman genome, and therefore makes it non-functional. This nucleotide wasdeveloped specifically for the study of cell targeting. When it istransfected, it is present in the cytoplasm and also penetrates into thecell nucleus.

Preparation of the Micelle/siRNA Complex:

3 μl of Block-It Fluorescent Oligo (20 μM), such as described in themanual “Block-It Transfection Kit” (Catalog ref. No.: 13750-070,Invitrogen), is mixed with 9 μl of cationic micelles E2 (octylβ-glucoside/cetyltriammonium bromide in the molar ratio of 5/1, 50 mM indistilled water).

Preparation of the Lipofectamine 2000/siRNA Mixture:

3 μl of Block-It Fluorescent Oligo (20 μM) is diluted in 50 μl ofOptiMEM (Invitrogen) and mixed with 3 μl of Lipofectamine 2000(Invitrogen) diluted in 12 μl of OptiMEM according to the supplier'sprotocol. The solution is incubated at ambient temperature for 20minutes to permit complexing of the liposomes and of the siRNA.

Preparation of Control Solutions:

3 μl of Block-It Fluorescent Oligo (20 μM) is diluted in 9 μl ofOptiMEM.

9 μl of cationic micelles E2 (octyl β-glucoside/cetyltriammonium bromidein the molar ratio of 5/1, 50 mM in distilled water) is diluted in 3 μlof OptiMEM.

The different mixtures and solutions are then deposited on differentsamples of EPISKIN epidermis and incubated under immersed conditions for48 hours at 37° C. in the presence of 5% CO2 in the differentiationmedium supplied with the EPISKIN kit. The samples are then frozen in amixture of dry ice and ethanol and cut into 5 μm sections by means of acryostat (MICROM HM560). The sections are mounted in Vectrashieldmounting medium (Vector Laboratories) containing 1.5 μg of DAPI andviewed under immunofluorescent conditions with filters appropriate forfluorescein and DAPI.

Observations:

Epidermis transfected on day 6: it is observed that, although thesolution of Block-It Fluorescent Oligo (Fig. I-1) or the mixture ofLipofectamine 2000 (Fig. I-2) does not permit penetration of Block-ItFluorescent Oligo beyond the stratum comeum being formed, the mixture ofE2 and Block-It Fluorescent Oligo permits penetration of the RNA duplexinto the epidermis (Figs. I-3, I-5, I-6). No non-specific marking isdetected when E2 is applied alone (Fig. I-4). The surprising characterof this observation is emphasized by the homogeneous presence of theBlock-It Fluorescent Oligo in all cells of the epidermis, whereas it isknown by the person skilled in the art that traditional transfection isefficient only in proliferating cells. In the reconstructed epidermis,only the basal cells proliferate, while the supra-basal cells becomedifferentiated.

Epidermis transfected on day 13: as on day 6, the solution of Block-ItFluorescent Oligo alone does not penetrate the stratum corneum (Fig.II-1). The mixture of E2 and Block-It Fluorescent Oligo permitspenetration of the fluorescent duplex into the different layers of thestratum corneum and into the different cells of the epidermis (Figs.II-2, II-3), albeit with lower efficiency and homogeneity than on day 6.

EXAMPLE 3 Effect of a Formulation of Octyl Glucoside and CTAB inDifferent Concentrations on the Mortality of HaCaT Cells

The effect of formula E2: 50 mM micelles of octyl glucoside/CTAB in 5/1molar ratio is achieved by incubation for 4 days according to thefollowing protocol on HaCaT cells (500 μl of medium seeded with 40,000cells per well, B-It: Block-It Fluorescent Oligo (Invitrogen).

40,000 HaCaT cells per well are seeded in duplicate in a 24-well platein 500 μl of complete DMEM+10% fetal calf serum.

1 μl of Block-It Fluorescent Oligo (Invitrogen) is diluted in 1 to 15 μlof micellar solution E2, composed of 50 mM of micelles of octylglucoside and CTAB in 5/1 molar ratio and added to the culture medium,bringing the final concentration of micellar solution to 0.1 to 1.5 mM.

The cells are then incubated for 4 days at 37° C., the medium for onesample of the duplicates being changed on day 1, after which they arephotographed.

The results obtained are presented in Figs. IIIA and IIIB.

No difference is observed between the cells subjected to the sametreatment with or without change of medium on day 1.

High mortality is observed for micellar solution concentrations belowthe CMC (approximately 1 mM), revealing the toxic effect of the vehiclein its free form, whereas good cell viability is observed above the CMC(1.5 mM).

The above written description of the invention provides a manner andprocess of making and using it such that any person skilled in this artis enabled to make and use the same, this enablement being provided inparticular for the subject matter of the appended claims, which make upa part of the original description and including a topical or ocularcomposition comprising at least one double stranded RNA oligonucleotide,characterized in that the double stranded RNA oligonucleotide isassociated with at least one cationic particle of size smaller than orequal to 1 μm, of zeta potential between 10 and 80 mV, chosen from amongsurfactant micelles, block polymer micelles, liposomes of nonionic andcationic surfactants, niosomes, oleosomes, particles of nanoemulsions,nanocapsules, organic particles or inorganic particles; the topical orocular use of the composition for cosmetic purposes, and a cosmetictreatment method by the application of the composition. Preferredembodiments of the invention similarly fully described and enabledinclude the composition characterized in that the cationic particle hasa size smaller than or equal to 500 nm, characterized in that thecationic particle has a size smaller than or equal to 300 nm,characterized in that the cationic particle is a micelle of nonionicamphiphilic surfactants and cationic surfactants, and/or characterizedin that the block copolymer micelle is chosen from among a micelle ofcationic amphiphilic block polymer, a micelle of nonionic amphiphilicblock polymer and of cationic amphiphilic block polymer and a micelle ofnonionic amphiphilic block polymer and of cationic surfactant.

Similarly enabled is a cosmetic treatment method comprising the topicalor ocular application composition characterized in that the cationicparticle has a size smaller than or equal to 500 nm, characterized inthat the cationic particle has a size smaller than or equal to 300 nm,characterized in that the cationic particle is a micelle of nonionicamphiphilic surfactants and cationic surfactants, characterized in thatthe block copolymer micelle is chosen from among a micelle of cationicamphiphilic block polymer, a micelle of nonionic amphiphilic blockpolymer and of cationic amphiphilic block polymer and a micelle ofnonionic amphiphilic block polymer and of cationic surfactant, andcharacterized in that the topical application is preceded by a skinpretreatment chosen from among stripping, chemical peeling, mechanicaldermabrasion, application of one or more solvents having a delipidatingeffect, or cleaning of the skin by a detergent foaming product, andcharacterized in that the topical application is followed by anocclusive post-treatment.

As used above, the phrases “selected from the group consisting of,”“chosen from,” and the like include mixtures of the specified materials.Terms such as “contain(s)” and the like as used herein are open termsmeaning ‘including at least’ unless otherwise specifically noted.

All references, patents, applications, tests, standards, documents,publications, brochures, texts, articles, etc. mentioned herein areincorporated herein by reference. Where a numerical limit or range isstated, the endpoints are included. Also, all values and subrangeswithin a numerical limit or range are specifically included as ifexplicitly written out.

The above description is presented to enable a person skilled in the artto make and use the invention, and is provided in the context of aparticular application and its requirements. Various modifications tothe preferred embodiments will be readily apparent to those skilled inthe art, and the generic principles defined herein may be applied toother embodiments and applications without departing from the spirit andscope of the invention. Thus, this invention is not intended to belimited to the embodiments shown, but is to be accorded the widest scopeconsistent with the principles and features disclosed herein.

As used herein, where a certain polymer is noted as being “obtainedfrom” or “comprising”, etc. one or more monomers (or monomer units) thisdescription is of the finished polymer material itself and the repeatingunits therein that make up, in whole or part, this finished product. Oneof ordinary skill in the art understands that, speaking precisely, apolymer does not include individual, unreacted “monomers,” but insteadis made up of repeating units derived from reacted monomers.

In this regard, the invention method and composition is preferably usedby subjects desirous of the benefits noted herein, subjects “in need of”these benefits. Such subjects are typically suffering from signs ofageing of the skin generally, from age-related hollowing of the faceand/or cheeks, or from age-related changes to the contour of the eyes,such as by self diagnosis or cosmetician or medical diagnosis, or are atrecognized and appreciated risk of developing such conditions and whouse the invention methods and compositions to combat these effects. Inthis regard, the invention process can be viewed as one for delaying theonset of the appearance of, and/or for reducing signs of, ageing of theskin.

Naturally, one using the invention as disclosed will use an amount ofthe invention composition effective to reduce the signs of ageing. Suchamount is inclusive of an amount of the compositions described herein atthe disclosed concentrations of active ingredients sufficient to coverthe area of the skin being treated in a single application, and ofcourse includes that amount applied upon repeated application, forexample on a daily basis over a course of days, weeks, etc. In apreferred embodiment the invention process includes multipleapplications of the invention composition to the area(s) of skin in needof attention.

1. A composition comprising at least one double stranded RNAoligonucleotide associated with at least one cationic particle of sizeless than or equal to 1 μm, having a zeta potential between 10 and 80mV, and selected from the group consisting of a surfactant micelle, ablock polymer micelle, a liposome of nonionic and cationic surfactants,a niosome, an oleosome, a nanoemulsion particle, a nanocapsule, anorganic particle, an inorganic particles and mixtures thereof; and atleast one ingredient suitable for topical and/or ocular application. 2.The composition according to claim 1, wherein the cationic particle hasa size less than or equal to 500 nm.
 3. The composition according toclaim 1, wherein the cationic particle has a size less than or equal to300 nm.
 4. The composition according to claim 1, wherein the cationicparticle is a micelle of nonionic amphiphilic surfactants and cationicsurfactants.
 5. The composition according to claim 1, wherein thecationic particle is a block copolymer micelle and which is selectedfrom the group consisting of a micelle of cationic amphiphilic blockpolymer, a micelle of nonionic amphiphilic block polymer and of cationicamphiphilic block polymer and a micelle of nonionic amphiphilic blockpolymer and of cationic surfactant.
 6. A method of providing a doublestranded RNA for a cosmetic purpose to an individual, comprisingapplying to at least one keratinous surface and/or at least one ocularsurface of the individual the composition of claim 1 in an amountsufficient to provide the double stranded RNA.
 7. The method accordingto claim 6, wherein the cationic particle has a size less than or equalto 500 nm.
 8. The method according to claim 6, wherein the cationicparticle has a size less than or equal to 300 nm.
 9. The methodaccording to claim 6, wherein the cationic particle is a micelle ofnonionic amphiphilic surfactants and cationic surfactants.
 10. Themethod according to claim 6, wherein the cationic particle is a blockcopolymer micelle and which is selected from the group consisting of amicelle of cationic amphiphilic block polymer, a micelle of nonionicamphiphilic block polymer and of cationic amphiphilic block polymer anda micelle of nonionic amphiphilic block polymer and of cationicsurfactant.
 11. The method according to claim 6, which furthercomprises, prior to applying the composition, at least one treatmentselected from the group consisting of chemical peeling, mechanicaldermabrasion, applying one or more solvents having a delipidatingeffect, and cleaning with a detergent foaming product.
 12. The methodaccording to claim 6, which further comprises, after applying thecomposition, an occlusive post-treatment.
 13. The method according toclaim 6, wherein the double stranded RNA penetrates the at least onekeratinous surface and/or at least one ocular surface after application.14. A method for inhibiting the expression of a protein in a cell of akeratinous and/or ocular material, comprising contacting the cell withat least one composition in an amount sufficient to inhibit expressionof the protein, wherein the composition comprises at least one doublestranded RNA oligonucleotide associated with at least one cationicparticle of size less than or equal to 1 μm, having a zeta potential offrom 10 to 80 mV and selected from the group consisting of a surfactantmicelle, a block polymer micelle, a liposome of nonionic and cationicsurfactants, a niosome, an oleosome, a nanoemulsion particle, ananocapsule, an organic particle, an inorganic particle, and mixturesthereof.
 15. The method according to claim 14, wherein the size of thecationic particle is less than or equal to 500 nm.
 16. The methodaccording to claim 14, wherein the size of the cationic particle is lessthan or equal to 300 nm.
 17. The method according to claim 14, whereinthe cationic particle comprises a micelle of nonionic amphiphilicsurfactants and cationic surfactants.
 18. The method according to claim14, wherein the cationic particle is a block copolymer micelle and whichis selected from the group consisting of a micelle of cationicamphiphilic block polymer; a micelle of nonionic amphiphilic blockpolymer and of cationic amphiphilic block polymer; a micelle of nonionicamphiphilic block polymer and of cationic surfactant; and a mixturethereof.